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G-LISA: Small G-protein Activation Assays

A revolutionary new way of doing Small G-protein Activation Assays!

The Rho family of small GTPases consists of at least 20 members, the most extensively characterized of which are the RhoA, Rac1 and Cdc42 proteins. In common with other small GTPases, the Rho proteins act as molecular switches that transmit cellular signals through downstream effector proteins by alternating between active GTP-bound and inactive GDP-bound states. The Rho family mediates a wide range of cellular responses, including cytoskeletal reorganization, regulation of transcription, membrane trafficking and apoptosis. Therefore, understanding the mechanisms that regulate activation and inactivation of the GTPases is of great biological significance and is a subject of intense investigation.

The traditional way of measuring the level of activation of a Rho protein has been to do so called pulldown activation assays. However, these methods are time consuming, require large amount of sample, tend to not be very consistent and are not suitable for high throughput screens. To circumvent these issues, Cytoskeleton Inc. has developed the G-LISA™ assay (patent pending).

The G-LISA Advantage

The G-LISA assay uses a 96-well plate coated with RBD domain of Rho-family effector proteins. The active GTP-bound form of the Rho-family protein, but not the inactive GDP-bound form, from a biological sample will bind to the plate. Bound active Rho-family protein is then detected by incubation with a specific primary antibody followed by a secondary antibody conjugated to HRP. The signal is then developed with OPD or chemiluminescence reagents.

These assays are simple, fast (<3 hours), require only small amounts of sample (5-25 µg cell protein), yield quantitative and accurate data and are suitable for high thoughput screens (see Table 1 in the gallery).

Kit content

The G-LISA kits contain all the reagents needed for the activation assay. All you need are your cell or tissue samples, a platform shaker and a microtiter plate compatible luminometer or spectrophotometer. The kit provides enough material for 96 assays. The affinity wells can be separated from each other, so you can run anywhere from 2-96 assays per experiment and each kit can be used for numerous separate experiments.

The G-LISA kits are available in either luminometric or colorimetric detection versions. The assays are identical except for the final detection step. In general: the luminometric assays are more sensitive, while the colorimetric assays have a slightly lower level of variance. See Table 2 in the gallery for comparison of the two versions of the kit.

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